Powassan virus nanobodies from camelid monomeric variable antibody domains elicited by natural infection

自然感染引起的骆驼单体可变抗体域的波瓦桑病毒纳米抗体

基本信息

批准号：
10043364
负责人：
MARGARET R MACDONALD
金额：
$ 25.43万
依托单位：
ROCKEFELLER UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-06-15 至 2022-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10043364
关键词：
Adopted Affinity Affinity Chromatography Alpaca Amino Acid Sequence Animals Antibodies Antibody Response Arboviruses Area Autoimmunity Bacteria Base Sequence Binding Biochemical Biological Biological Assay Biological Products Black-legged Tick Bone Marrow Camels Canada Cessation of life Chemicals Clinical Clinical Trials Communicable Diseases Communities Computer software DNA Library Data Development Diagnostic Disease E protein Encephalitis Environment Enzyme-Linked Immunosorbent Assay Epidemiology Escherichia coli Exposure to Family Fc domain Flaviviridae Flavivirus Future Goals Great Lakes Region High-Throughput Nucleotide Sequencing Human Immunity Immunization Immunize In Vitro Individual Infection Ixodes Laboratories Life Llama Longevity Lymphocyte Magic Malignant Neoplasms Mammals Massachusetts Medical Medical Research Methods Modeling Molecular Monoclonal Antibodies Monoclonal Antibody Therapy Mus Neurologic Outcome Penetrance Peptides Population Powassan virus Prevalence Proteins Publishing RNA Reagent Recombinants Recovery Reporter Research Role Russia Safety Series Serological Solubility Specificity Structure Supportive care System Tertiary Protein Structure Testing Therapeutic Therapeutic Agents Therapeutic Uses Ticks Tissues Translating Treatment Efficacy United States Vaccination Variant Viral Antigens Virion Virus Virus Diseases base cancer therapy cost cross reactivity disorder control env Gene Products experimental study immunogenicity in silico liquid chromatography mass spectrometry medical attention member mosquito-borne pathogen nanobodies neutralizing antibody neutralizing monoclonal antibodies nonhuman primate novel novel virus pathogen programs protein E response success tick-borne tool virology virus envelope

项目摘要

Powassan virus (POWV), a tick-borne flavivirus endemic to the USA, Canada, and Russia, has steadily increased in prevalence over the past decade. In a subset of cases, it leads to severe, often fatal, encephalitic disease. Furthermore, following recovery, long-term debilitating neurological sequelae occur in ~50% of individuals. No virus-specific therapeutic options currently exist, restricting treatment to supportive care. Monoclonal antibodies have been used clinically since 1986 for cancer, autoimmunity and infectious disease control and neutralizing monoclonal antibody therapy for POWV is one potential treatment approach. Nanobodies, or the variant domains (VHH) of heavy-chain only antibodies (HcAb) found in camelid species, have recently been adopted by the medical research community, with several currently in clinical trials for a variety of diseases and diagnostics. Nanobodies have low immunogenicity, high specificity, and exceptional stability and solubility, as well as demonstrated superior efficacy, safety, and utility, making them strong candidates for future therapeutics. Serological data suggests that many alpacas and llamas in the NE USA may be exposed to POWV during their lifespan, and therefore likely harbor neutralizing antibodies. This study combines virological, molecular biochemical, and epidemiological approaches to identify and characterize novel POWV nanobodies derived from naturally infected and immunized camelid species. In Aim 1, total sera from alpacas and llamas will be screened for POWV neutralization activity. Positive animals will be immunized with recombinant envelope protein in order to boost antibody responses. From animals whose sera show the highest neutralization potential, POWV- specific HcAb populations will be purified by affinity chromatography and the 15 kDa VHH domains isolated. Bone marrow will also be extracted from these same serologically positive animals and the VHH loci will be amplified by PCR. Utilizing a two-pronged identification strategy, VHH protein pools and VHH loci will be characterized by LC/MS and MiSeq high-throughput sequencing, respectively. Loci sequences will then be translated in silico and matched to the MS data using our program Llama-Magic, providing complete amino acid sequences for POWV-specific nanobodies. In Aim 2, recombinant nanobodies will be expressed in E. coli, purified, and tested in vitro for POWV binding and neutralization as well as cross reactivity to other flaviviruses. This will yield a panel of potent POWV-specific nanobodies that will be useful for research, diagnostic and therapeutic purposes. Future experiments could explore their therapeutic efficacy using murine and non-human primate models of POWV encephalitis.

在过去的十年中，美国，加拿大和俄罗斯特有的tick传播的黄病毒（Powv）是美国，加拿大和俄罗斯的特有tick虫的流行病。在一部分情况下，它会导致严重的，通常是致命的脑病。此外，恢复后，在约50％的个体中发生了长期衰弱的神经后遗症。目前没有病毒特异性的治疗选择，将治疗限制为支持护理。自1986年以来，单克隆抗体已在临床上用于癌症，自身免疫性和感染性疾病控制和中和POWV中和单克隆抗体治疗是一种潜在的治疗方法。医学研究界最近采用了纳米型或仅在骆驼物种中发现的重链抗体（HCAB）的变异域（VHH），目前正在进行一些针对各种疾病和诊断的临床试验。纳米生物的免疫原性，高特异性和卓越的稳定性和溶解度，并且具有出色的功效，安全性和实用性，使其成为未来治疗剂的强大候选者。血清学数据表明，美国东北部的许多羊驼和美洲狮在其寿命中可能会暴露于POWV，因此很可能避开中和抗体。这项研究结合了病毒学，分子生化和流行病学方法，以识别和表征源自自然感染和免疫性骆驼物种的新型POWV纳米体。在AIM 1中，将筛选来自羊驼和美洲驼的总血清以进行POWV中和活性。为了增强抗体反应，将用重组包膜蛋白免疫阳性动物。从血清表现出最高中和潜力的动物中，特定于特定的HCAB种群将通过亲和力色谱和分离的15 kDa VHH结构域纯化。骨髓还将从这些同一血清学阳性动物中提取，VHH基因座将通过PCR扩增。通过利用两种普通的识别策略，VHH蛋白库和VHH基因库将分别以LC/MS和Miseq高通量测序为特征。然后，将使用我们的程序Llama-Magic将基因座序列翻译成有机硅，并与MS数据匹配，从而为POWV特异性纳米型提供完整的氨基酸序列。在AIM 2中，将在大肠杆菌中表达重组纳米体，并在体外进行了纯化和测试，以实现POWV结合和中和以及对其他Flavivivires的交叉反应性。这将产生一系列有效的POWV特异性纳米化合物，这些纳米体将对研究，诊断和治疗目的有用。未来的实验可以使用POWV脑炎的鼠和非人类灵长类动物模型来探索其治疗功效。